Renal tubular epithelial cell (RTC) apoptosis causes tubular atrophy, a hallmark of chronic renal disease. The original term for apoptosis was "shrinkage necrosis", based upon descriptions of reduced cytoplasmic volume. Apoptotic cells also develop cytosolic acidification, which promotes caspase activation. Preliminary data demonstrate that the plasma membrane Na+/H+ transporter, NHE1, promotes RTC survival by defending cell volume and pHi through Na+/H+ exchange. In addition, apoptotic or osmotic stress activates a signaling cascade that links the NHE1 cytosolic domain to ezrin, radixin, moesin (ERM) proteins, which tether NHE1 to cortical actin, followed by downstream activation of the cell survival kinases, PIS kinase and Akt. Published reports and preliminary data also implicate Rho-dependent kinase (ROCK) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the pathway. Furthermore, a robust death stimulus is associated with caspase-3 degradation of NHE1, cell shrinkage and cytosol acidification. In vivo, NHE1- deficient mice demonstrate increased RTC apoptosis following renal disease induction with adriamycin or streptozotocin. The hypothesis is that NHE1 activation stimulates ROCK activity and ERM recruitment to the NHE1 cytoplasmic domain, to form a cell survival signalplex within a PIP2-rich plasma membrane microenvironment. Tubular atrophy and renal disease progression require NHE1 inactivation due to caspase cleavage of the NHE1 cytoplasmic tail. The hypothesis will be pursued with the following specific aims: (1) To characterize assembly of the NHE1-regulated cell survival signalplex, which is activated by osmotic/apoptotic stress, using biochemical and immunolocalization techniques, (2) To determine the role of NHE1 as a caspase substrate, by identification of the caspase(s) that cleave NHE1, by mapping the NHE1 caspase cleavage sites, and by testing the effect of cleavage-resistant NHE1 mutant expression on cell survival, and (3) To test the role of NHE1 in RTC survival in vivo, kidney phenotypes will be determined in NHE1-deficient vs. control mice following induction of progressive renal diseases. The proposed experiments will prove that NHE1 is a critical cell survival factor and caspase target. Establishing NHE1 as an arbiter of cell survival or death would warrant the design of therapeutic strategies to stabilize the NHE1- regulated survival signalplex and/or inhibit caspase cleavage of NHE1.